Enzymatic and crystallographic characterization of archaeal tRNA splicing endonuclease

Abstract

RNA splicing endonuclease, which is conserved in Eukarya and Archaea, removes introns from eukaryotic nuclear tRNA and archaeal all RNA species. There are three types of subunit composition in archaeal RNA splicing endonucleases, namely ¿ <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> (homodimer in some Euryarchaea), ¿ <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</sub> (homotetramer in other Euryarchaea) and (¿ß) <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> (heterotetramer in the Crenarchaea) types. In Crenarchaea, introns in precursor tRNA (pre-tRNA) molecules are found not only in anticodon arm but also in several other regions in tRNA such as D- and T-loops, variable region, and aminoacyl stem. These introns have a consensus bulge-helix-bulge motif. Crenarchaeal RNA splicing endonuclease can remove introns in such non-canonical sites. However, the broad cleavage site-specificity of the enzyme remains exclusive. Here, we report the cleavage activity of recombinant RNA splicing endonuclease from hyperthermophilic crenarcheon Aeropyrum pernix (APE) by using the pre-tRNA <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Thr (UGU)</sup> and pre-tRNA <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Thr (CGU)</sup> , in which introns are located in anticodon and D-loops, respectively. Furthermore we report that APE RNA splicing enzyme crystallizes in space group P3(1) with two or three heterotetramers in an asymmetric unit. An X-ray diffraction data set has been collected to 2.8 A¿ resolution. Structural determination is now underway.